Translation, the mRNA-directed synthesis of proteins, occurs in three distinct steps: initiation, elongation and termination. Translation initiation is a complex process in which the two ribosomal subunits and methionyl tRNA (Met-tRNAi) assemble on a properly aligned mRNA to commence chain elongation at the AUG initiation codon. The established scanning mechanism for initiation involves the formation of a ternary complex among eukaryotic initiation factor 2 (eIF2), GTP and Met-tRNAi. The ternary complex recruits the 40S ribosomal subunit to form the 43S pre-initiation complex. This complex recruits mRNA in cooperation with other initiation factors such as eukaryotic initiation factor 4E (eIF4E), which recognizes the 7-methyl-guanidine cap (m-7GTP cap) in an mRNA molecule and forms the 48S pre-initiation complex. Cap recognition facilitates the 43S complex entry at the 5′ end of a capped mRNA. Subsequently, this complex migrates linearly until it reaches the first AUG codon, where a 60S ribosomal subunit joins the complex, and the first peptide bond is formed (Pain (1996) Eur. J. Biochem., 236:747-771). After each initiation, the GTP in the ternary complex is converted to GDP. The eIF2.GDP binary complex must be converted to eIF2.GTP by the guanidine exchange factor, eIF2B for a new round of translation initiation to occur. Inhibition of this exchange reaction by phosphorylation of eIF2α reduces the abundance of the ternary complex and inhibits translation initiation. Forced expression of non-phosphorylatable eIF2α or Met-tRNAi causes transformation of normal cells (Marshall (2008) Cell 133:78; Berns (2008) Cell 133:29). In contrast, pharmacologic agents that restrict the amount of eIF2.GTP.Met-tRNAi ternary complex inhibit proliferation of cancer cells in vitro and tumors in vivo (Aktas (1998) Proc. Natl. Acad. Sci. U.S.A. 95:8280), Palakurthi (2000) Cancer Res. 60:2919, Palakurthi (2001) Cancer Res. 61: 6213). These findings indicate that more potent and specific agents that reduce amount of ternary complex are potent anti-cancer agents.
Several features of the mRNA structure influence the efficiency of its translation. These include the m-7GTP cap, the primary sequence surrounding the AUG codon and the length and secondary structure of the 5′ untranslated region (5′ UTR). Indeed, a moderately long, unstructured 5′ UTR with a low G and C base content seems to be optimal to ensure high translational efficiency. Surprisingly, sequence analysis of a large number of vertebrate cDNAs has shown that although most transcripts have features that ensure translational fidelity, many do not appear to be designed for efficient translation (Kozak (1991) J. Cell. Biol., 115:887-903). Many vertebrate mRNAs contain 5′ UTRs that are hundreds of nucleotides long with a remarkably high GC content, indicating that they are highly structured because G and C bases tend to form highly stable bonds. Because highly structured and stable 5′ UTRs are the major barrier to translation, mRNAs with stable secondary structure in their 5′ UTR are translated inefficiently and their translation is highly dependent on the activity of translation initiation factors.
mRNAs with complex, highly structured 5′ UTRs include a disproportionately high number of proto-oncogenes such as the G1 cyclins, transcription and growth factors, cytokines and other critical regulatory proteins. In contrast, mRNAs that encode globins, albumins, histones and other housekeeping proteins rarely have highly structured, GC-rich 5′ UTRs (Kozak (1994) Biochimie, 76; 815-21; Kozak (1999) Gene, 234:187-208). The fact that genes encoding for regulatory but not for housekeeping proteins frequently produce transcripts with highly structured 5′ UTRs indicates that extensive control of the expression of regulatory genes occurs at the level of translation. In other words, low efficiency of translation is a control mechanism which modulates the yield of proteins such as cyclins, mos, c-myc, VEGF, TNF, among others, that could be harmful if overproduced.
Translation initiation is a critical step in the regulation of cell growth because the expression of most oncogenes and cell growth regulatory proteins is translationally regulated. One approach to inhibiting translation initiation has recently been identified using small molecule known as translation initiation inhibitors. Translation initiation inhibitors such as clotrimazole (CLT) inhibit translation initiation by sustained depletion of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores activates “interferon-inducible” “double-stranded RNA activated” protein kinase (PKR) which phosphorylates and thereby inhibits the α subunit of eIF2. Since the activity of eIF2 is required for translation initiation, its inhibition by compounds such as CLT reduces the overall rate of protein synthesis. Because most cell regulatory proteins are encoded for by mRNAs containing highly structured 5′ UTRs, they are poorly translated and their translation depends heavily on translation initiation factors such as eIF2 and eIF4. Therefore, inhibition of translation initiation preferentially affects the synthesis and expression of growth regulatory proteins such as G1 cyclins. Sequential synthesis and expression of G1 cyclins (D1, E and A) is necessary to drive the cell cycle beyond the restriction point in late G1. Thus, the decreased synthesis and expression of G1 cyclins resulting from CLT-induced inhibition of translation initiation causes cell cycle arrest in G1 and inhibits cancer cell and tumor growth (Aktas et al. (1998) Proc. Natl. Acad. Sci. USA, 95:8280-8285, incorporated herein by reference in its entirety for all purposes).
Like CLT, the n−3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) depletes internal calcium stores, and exhibits anti-carcinogenic activity. Unlike CLT, however, EPA is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), a fatty acid-activated transcription factor. Although EPA and other ligands of PPARγ, such as troglitazone and ciglitazone, inhibit cell proliferation, they do so in a PPARγ-independent manner (Palakurthi et al. (2000) Cancer Research, 60:2919; and Palakurthi et al. (2001) Cancer Research, 61:6213, incorporated herein by reference in their entirety for all purposes).